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Update on Recombinational Cloning with Plant Gateway Vectors

Recombinational Cloning with Plant Gateway Vectors¹

Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department of Molecular Genetics, Ghent University, 9052 Ghent, Belgium

The study of biological systems relies to a large extent on DNA cloning technologies enabling the analysis of recombinant genes through transgenic research. In this context, the advent of recombinational cloning methods was a significant progress because DNA fragments can now be assembled regardless of their sequence. In particular, the Gateway system was designed to join fragments in a predefined order, orientation, and reading frame. The recent development of transformation vectors and large-scale clone resources amply demonstrate that plant researchers have adopted the Gateway platform and that it will remain an important asset in projects requiring systematic cloning, modular assembly, and expression in various contexts.

Agrobacterium tumefaciens binary vectors are widely used for plant transformation. They vary in size, origin of replication, bacterial selectable markers, T-DNA borders, and overall structure. Binary vectors are cumbersome to handle in conventional cloning schemes involving DNA restriction and ligation reactions, and substantial efforts have been invested in the creation of smaller vectors with a choice of unique restriction sites within the T-DNA region (Hajdukiewicz et al., 1994; Hellens et al., 2000a; Goderis et al., 2002; Tzfira et al., 2005; http://www.cambia.org/). But the recent introduction of robust site-specific recombinational cloning methods has greatly facilitated the construction of expression units in a large variety of in vivo and in vitro systems (Marsischky and LaBaer, 2004). In particular, the Gateway technology developed originally by researchers at Life Technologies, Inc. (Hartley et al., 2000), and now commercialized by Invitrogen, has been endorsed by a large community and compatible vectors have been created for most applications requiring the creation of recombinant DNA molecules. This review gives a summary of the site-specific Gateway recombinational cloning system and presents related vectors generated by different plant research laboratories.

	BASIC PRINCIPLES OF GATEWAY RECOMBINATIONAL CLONING

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The Gateway system takes advantage of the site-specific recombination reactions enabling the bacteriophage to integrate and excise itself in and out of a bacterial chromosome (for review, see Katzen, 2007). Gateway protocols rely essentially on the BP and LR clonase reactions (Hartley et al., 2000). The BP reaction is catalyzed by the BP Clonase II enzyme mix that consists of the phage integrase and the integration host factor (Fig. 1A ). The BP clonase mix transfers a DNA fragment of interest, for example a PCR product, flanked by two attB sites into a donor vector (pDONR) carrying two attP sites. After recombination of the matching attB and attP sites, the DNA fragment is inserted into the donor backbone, resulting in an entry clone (pENTR), and is flanked by two attL sites. Entry clones can also be assembled by restriction and ligation of DNA fragments in vectors in which multiple cloning sites are flanked by attL sites. In most cases, entry clones are by themselves not directly useful because attL sites are too long (96 bp) to be placed as spacers between sequences of interest. In comparison, engineered attB sites are only 21 to 25 bp in length and have been designed without translation initiation or stop codon (Hartley et al., 2000). Entry clones are key substrates in the LR reaction that is catalyzed by the LR Clonase II enzyme mix that consists of integrase, integration host factor, and the phage excisionase (Fig. 1B). The LR clonase mix transfers the DNA fragment of interest flanked by two attL sites (in the entry clone) into a destination vector (pDEST) carrying two attR sites. After recombination of the matching attL and attR sites, the DNA fragment of interest is inserted into a novel expression clone (pEXPR) and again flanked by attB sites. Donor vector (pDONR), entry clone (pENTR), destination vector (pDEST), and expression clone (pEXPR) are terms adopted by Gateway users to distinguish the input and output plasmids in clonase reactions (Fig. 1).

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Schematic representation of att sites and Gateway recombination reactions. A, In a BP clonase reaction, attB sites (in a PCR product or plasmid) recombine with the matching attP sites of a donor vector (pDONR) to yield attL sites in a novel entry vector (pENTR) and attR sites in a byproduct. B, In a LR clonase reaction, attL sites in an entry vector (pENTR) recombine with the matching attR sites of a destination vector (pDEST) to yield attB in a novel expression vector (pEXPR) and attP in a byproduct. C, In a single MultiSite LR clonase reaction, the compatible att sites carried by entry clones originating from independent BP clonase reactions (in this example pENTR L4-fragment1-R1, pENTR L1-fragment2-L2, and pENTR R2-fragment3-L3) and by a MultiSite destination vector (for example pDEST R4-R3) recombine to yield a single contig in which the DNA fragments of interest are separated by short attB sites. Blue boxes, DNA fragments of interest assembled in BP and LR clonase reactions; black box with vertical white stripe, attB sites, also at the core of the attP, attL, and attR sites; yellow box, portion of the attP and attL sites; red box, portion of the attP and attR sites. B1 to B4, attB1 to attB4 sites; L1 to L4, attL1 to attL4; R1 to R4, attR1 to attR4.

A given destination vector intended for a particular functional assay can be recombined with any sequence captured in a compatible entry clone. Conversely, the same entry clone (such as an open reading frame [ORF] entry clone) can be recombined with many different destination vectors. The resulting expression clones are the constructs used to test gene functions, for example after transformation into plants.

Typically, the desired plasmids are created by in vitro recombination, transformed in Escherichia coli strains, and segregated from other reaction by-products and input vectors through appropriate antibiotic selection and counterselection based on the ccdB (control of cell death) gene (Hartley et al., 2000). Importantly, the backbones of the plant binary T-DNA destination vectors must contain different bacterial antibiotic resistance markers from the donor (and entry) vectors (Table I ). Since most donor vectors used in BP reactions code for kanamycin resistance, expression clones resulting from LR reactions should carry other bacterial selectable markers. Some T-DNA vector series encoding kanamycin resistance, such as pGreen (Hellens et al., 2000b) and most pCambia accessions (http://www.cambia.org/), are not ideally suited for the construction of destination vectors. They require either linearization of the entry clone prior to LR reaction or the use of alternative donor vectors. Other factors are important to consider when creating or choosing a binary destination vector: the plant-selectable marker carried within the T-DNA, especially when combining different transgenic loci in the same plants; its location in the T-DNA preferably close to the left border because the transfer from bacterium to plant starts with the right border and the gene of interest should be integrated before the selectable marker (Hellens et al., 2000a); and its regulatory sequences, for example avoiding the strong cauliflower mosaic virus (CaMV) 35S promoter that alters the level and pattern of activity of adjacent promoters (Zheng et al., 2007).

	DESTINATION VECTORS FOR THE ANALYSIS OF PLANT GENE FUNCTION

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Ectopic Overexpression

In destination vectors designed for gene overexpression, a standard Gateway cassette (attR1-ccdB-attR2) is placed downstream of the CaMV 35S promoter and upstream of a standard terminator (Tables I, nos. 1–6 and II, no. 1). A similar configuration can be obtained with MultiSite Gateway binary vectors for two or three fragment recombination (Table I, nos. 84–88) with the added advantage that any strong, inducible, or tissue-specific promoter captured in an entry vector (attL4-promoter-attR1) can be fused with any gene of interest present in another entry clone (attL1-gene-attL2). Already, a collection of sequence-validated promoters (including CaMV 35S, Cassava vein mosaic virus [CsVMV], A. tumefaciens nopaline synthase [nos], maize [Zea mays] ubiquitin [ubi], and A. rhizogenes rolD) is available for MultiSite LR reaction that is complemented with terminator entry clones (attR2-terminator-attL3) including CaMV 35S, nos, octopine synthase, and gene7 (Karimi et al., 2007).

For certain experiments gene expression has to be induced at a chosen time, for example to characterize loss- or gain-of-function mutations that cause embryo lethality. Vectors that have been constructed for the inducible transcriptional activation of a gene of interest are described below. An alternative system has been devised for misexpression restricted to clonal sectors. In this configuration, the gene of interest is cloned in a Gateway cassette placed downstream of the 35S CaMV promoter, but separated from it by a spacer containing a gene coding for the ENHANCED GFP (EGFP) flanked by loxP sites. Another transgene carried within the same T-DNA expresses the CRE recombinase under the control of the promoter of the heat shock protein gene HSP18.2 of Arabidopsis (Arabidopsis thaliana). To avoid any unwanted CRE activity, the CRE enzyme is fused to the mammalian glucocorticoid receptor (GR) domain that localizes the hybrid protein into the nucleus only in the presence of dexamethasone (DEX). Thereby, the gene of interest is constitutively activated upon combined heat and DEX treatments, resulting in the excision of the EGFP spacer by CRE recombination of the two loxP sites. The activation sectors are marked by the absence of the GFP fluorescence (Joubès et al., 2004; Table I, no. 66). CRE-GR-mediated activation was used successfully in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, but not in Arabidopsis (L. De Veylder, personal communication). A similar system, proven to be reliable in Arabidopsis root tissues, was constructed in which CRE activity does not require DEX treatment (De Schutter et al., 2007; Table I, no. 67). Independently, binary destination vectors have also been created for cloning any promoter of choice upstream of the CRE ORF (Marjanac et al., 2007; Table I, no. 89).

cis-Regulatory Sequence Analysis, Translational Fusion, and Subcellular Localization

The transcriptional activity of a promoter can be characterized by the temporal and spatial expression patterns of a reporter protein. In such studies, the promoter sequence must first be cloned upstream of the corresponding reporter gene. Alternatively, the presence of a protein of interest can be monitored if it carries a tag at its C or N terminus, after transcription under the control of its own promoter (Tables I, nos. 10–13,17–20 and II, no. 2) or of a heterologous strong promoter, such as CaMV 35S (Tables I, nos. 29–63 and II, nos. 3–18). When the added domain in a translational fusion codes for a fluorescent protein or an epitope tag, the protein of interest can be localized subcellularly by microscopic analysis of living or fixed cells (Citovsky et al., 2006).

Binary destination vectors have been designed in which a standard Gateway cassette (attR1-ccdB-attR2) precedes the ORF coding for an enzyme (luciferase [LUC] or GUS), a fluorescent protein (GFP, yellow fluorescent protein [YFP], cyan fluorescent protein [CFP], or red fluorescent protein [RFP]), with or without a nuclear localization signal, a purification tag (polyhistidine [6xHis]), an epitope tag (hemagglutinin [HA], FLAG, c-Myc, or AcV5), or a combination of these (Table I, nos. 9–20). The same final configuration can be obtained via MultiSite Gateway recombination (Table I, no. 21). In some of these constructs, the spacer at the 3' end of the inserted fragment (with regard to transcription) is structured to enable translational fusion so that a protein of interest encoded in a DNA sequence with its native promoter can be expressed with an additional domain at its C terminus (Table I, nos. 11–20). This type of arrangement is necessary to study cis-regulatory sequences located in introns or exons.

The MultiSite Gateway system can also be used to assemble a promoter of choice (attL4-promoter-attR1), the selected reporter tag (attL1-reporter-attL2 or attR2-reporter-attL3), and the gene coding for the protein of interest (attR2-gene-attL3 or attL1-gene-attL2, respectively) in a compatible destination vector (Table I, no. 88). Numerous sequence-validated reporter entry clones are available for such LR cloning (fluorescence tags [GFP, CFP, YFP, or RFP], epitope tags [Myc, HA, or FLAG], or enzyme tags [GUS or LUC]) of ORFs expressing N- or C-terminal fusions (Karimi et al., 2007).

In all Gateway constructs expressing translational fusions, the tags, genes, ORFs, or cDNAs captured in the entry clones are assembled in the same frame register and are separated by attB sites coding for eight amino acids (e.g. XAFLYKVX for attB2 and XTLLYIVX for attB3). Some reports argue that the spacers encoded in attB sites are better than those encoded in multiple restriction cloning sites, or vice versa. In our hand, the success or failure of a functional assay involving translational fusion does not depend on a particular cloning strategy, but instead varies on a case-by-case basis.

The ORF structure in the entry clones needs to be carefully considered when constructs are designed for expression of tagged proteins. For example, it might be preferable to place a tag at the N terminus of the characterized protein when posttranslational modifications occur at or near its C terminus that are essential for function. However, at least one-quarter of the Arabidopsis protein coding sequences include a predicted N-terminal targeting signal peptide (Millar et al., 2006) that might not be functional when fused to an additional tag or might cause the tag to be clipped off the processed protein. On the other hand, the addition of a C-terminal tag requires removal of the original stop codon from the ORF. Depending on downstream applications, ORF entry clones might have to be generated both in open (without stop codon) or closed (with native stop codon) configurations (Underwood et al., 2006).

Two-Component Systems

Two-component systems have been developed for conditional gene activation or silencing. They combine an activator (or driver) locus that codes for an artificial transcription factor expressed in restricted tissues, at precise developmental times, or upon environmental or chemical induction, and a responder (or recipient) locus in which an artificial promoter controls the transcription of a gene under investigation, when activated by the transcription factor. Building these loci separately is advantageous in research projects requiring the combination of multiple activator and responder transgenes, either by consecutive transformation or via crosses.

The XVE plant two-component system has been developed for the chemical induction of gene expression by the estrogen hormone and adapted to the Gateway format. When applied, estrogen binds to and activates the XVE chimeric transcription factor that consists of three domains: DNA binding (LexA), transcriptional activation (VP16), and estrogen receptor (Zuo et al., 2000). In the basic activator vector (Table I, no. 68), any promoter or enhancer sequence (attL1-promoter-attL2) can be cloned upstream of the XVE ORF (Brand et al., 2006). In another vector, the XVE transgene is located in the same T-DNA as a GUS responder gene that marks the domain of XVE activation (Table I, no. 72). Responder vectors (Table I, nos. 69–71) include an XVE-responsive promoter upstream of a Gateway cassette (attR1-ccdB-attR2) for XVE-mediated transcriptional induction of any gene of interest, linked to a bacterial ampicillin resistance gene and a ColE1 origin of replication that can be used for plasmid rescue (Table I, nos. 69–71).

Elements of another two-component system have been formatted for MultiSite Gateway cloning (Karimi et al., 2007). It is based on either one of two artificial transcription factors consisting of the yeast (Saccharomyces cerevisiae) GAL4 activation domain fused to the bacterial LacI repressor DNA-binding domain (Moore et al., 1998): LhG4 that is constitutively active (Rutherford et al., 2005) and LhGR2 that contains the GR domain and is inducible by DEX treatment (Craft et al., 2005). LhG4 and LhGR2 bind to lac operator (pOp) sequences. LhG4 and LhGR2 ORFs as well as a pOp promoter have been captured in entry clones. Thereby, both the driver and recipient transgenes at the core of this two-component system can be assembled via MultiSite cloning (Karimi et al., 2007).

Gene Silencing

Following the introduction of plant transformation technology, plant gene silencing was achieved by transcribing at high level homologous cDNAs from transgenic loci, via a process called cosuppression (Jorgensen et al., 1998). Gateway overexpression vectors may still be used to this end. In addition, long double-stranded RNAs have been shown to trigger gene silencing via RNA interference (RNAi; Small, 2007). In particular, hairpin RNA (hpRNA) molecules that initiate the synthesis of small interfering RNA, are very potent RNAi inducers in plant cells (Smith et al., 2000). Although it is not trivial to design a locus for the transcription of mRNAs corresponding to the two opposite strands of the same DNA segment, an interesting twist of the Gateway LR recombination made this step straightforward (Wesley et al., 2001). The sequence targeted for silencing, captured in an entry clone (attL1-target-attL2), is transferred in a single LR clonase reaction in a destination vector carrying two independent Gateway cassettes separated by an intron spacer (attR1-ccdB-attR2-intron-attR2-ccdB-attR1). The Gateway cassettes are identical except that their attR1 and attR2 recombination sites are inverted with respect to one another, so that the two copies of the target sequence are positioned head to head in the resulting hpRNA expression clone.

Several binary destination vectors have been constructed according to this scheme. They differ in backbone, structure of the intron spacer, and promoter controlling the transcription of the hpRNA (Table I, nos. 22–27). Notably, a vector has been created for the chemical (DEX) induction of RNAi, taking advantage of the pOp6/LhGR two-component system (Rutherford et al., 2005; Table I, no. 28). Alternatively, the assembly of vectors for the expression of hpRNAs under the control of any promoter of choice can also be facilitated by a two-step procedure only involving MultiSite Gateway cloning, as described elsewhere (A.I. Fernandez, M. Karimi, M. Jones, Z. Amsellem, A. Sicard, A. Czerednik, G. Angenent, D. Grierson, S. May, C. Rothan, G. Seymour, Y. Eshed, and P. Hilson, unpublished data). The double LR clonase reaction is most efficient with destination vectors in which the sequence between the attR sites (including the ccdB counterselectable marker) is inserted as a direct repeat (Table I, nos. 24–27) instead of an inverted repeat (Helliwell and Waterhouse, 2003). A caveat of RNAi approaches based on the constructs described above is that the silencing hpRNA includes transcribed double-stranded attB1 and attB2 sites that are sufficient to knock down expression of an independent transgene including the same Gateway sites (R. Vanderhaeghen and P. Hilson, unpublished data).

Recently, artificial microRNAs (amiRNAs) have also been used to silence plant genes (Alvarez et al., 2006; Schwab et al., 2006). These amiRNAs are engineered from endogenous microRNA precursors (such as MIR319a) in which the two key short nucleotide sequences that form the miRNA—defining which transcripts are cleaved and undergo subsequent degradation—are replaced by sequences chosen to be as specific as possible to the target transcript(s) (Schwab et al., 2005). Silencing via amiRNA seems highly specific and can be induced under the control of specific promoters. Interestingly, the original method described by Schwab et al. (2006) to synthesize an amiRNA in a single overlap PCR that joins three DNA segments can be adapted to the Gateway cloning framework by simple addition of the attB1 and attB2 recombination sites (replacing restriction sites) at the extremity of the outermost primers. The resulting amiRNA precursor amplicon can be captured in an entry clone (attL1-amiRNA-attL2), then transferred into a destination vector as any other gene, via a standard or MultiSite LR clonase reaction, for transcription in different tissues, at various developmental times, or upon induction. The addition of the attB sites at the flanks of an amiRNA sequence does not impair its silencing ability (A.I. Fernandez, M. Karimi, M. Jones, Z. Amsellem, A. Sicard, A. Czerednik, G. Angenent, D. Grierson, S. May, C. Rothan, G. Seymour, Y. Eshed, and P. Hilson, unpublished data; F. Coppens, R. Vanderhaeghen, and G.T.S. Beemster, personal communication).

Virus-induced gene silencing (VIGS) has also been used to knock down gene expression in plants (Burch-Smith et al., 2004; Robertson, 2004). In VIGS experiments, plants are inoculated with a viral vector carrying a fragment derived from a host gene. VIGS screens have the advantage to bypass the need to create stable transformants for each tested target gene, but are limited when viral infection yields symptoms on its own. Also, infection is generally confined to certain tissues, but this restriction might be mitigated by the spread of the silencing signals. Several VIGS vectors have been modified to be compatible with Gateway cloning (Tables I, no. 78 and II, no. 24).

Genomic Fragment Recombination

Key functional tests, such as the complementation of mutant alleles, require the reintroduction into selected genotypes of an intact genomic DNA region. Several binary destination vectors have been designed for such simple genomic fragment recombinations (Table I, nos. 79–83). In some of these, the T-DNA also incorporates a visible reporter (Table I, no. 80), enabling the distinction between transformed and untransformed cells mixed in chimeric tissues, such as hairy roots formed upon A. rhizogenes-mediated transformation (Van de Velde et al., 2003; Baranski et al., 2006).

Protein-Protein Interaction

The interaction between polypeptides in living cells can be determined with methods that require the production of proteins tagged via in-frame translational fusion. For tandem affinity purification (TAP), the tag codes for a domain that associates reversibly and with high affinity to one or multiple ligands generally fixed on solid beads and mixed with cell extracts (Puig et al., 2001). After elution of unbound materials, the composition of the tagged-purified fraction, i.e. the partners associated in the purified heterocomplex, can be analyzed by mass spectrometry. For bimolecular fluorescence complementation, two hybrid proteins are produced, each carrying one inactive half of a fluorescent moiety (GFP or YFP). When the hybrids interact, they reconstitute a functional fluorescent protein that is visualized microscopically. Detection of interaction between two proteins via fluorescence resonance energy transfer relies on the nonradiative energy transfer between polypeptides, each carrying a different fluorescent tag. In such configurations, close proximity between the donor and acceptor chromophores (such as CFP and YFP, respectively) translates into a decrease in the fluorescence lifetime of the donor moiety.

TAP, bimolecular fluorescence complementation, and fluorescence resonance energy transfer have already been implemented in plant cells through Gateway destination vectors for the production of proteins with various TAP tags (Brown et al., 2006; Van Leene et al., 2007), truncated fluorescent proteins (Walter et al., 2004), or CFP and YFP tags (Huang et al., 2006; Tonaco et al., 2006; Tables I, nos. 73–77 and II, nos. 19 and 20).

Finally, two-hybrid systems in which the protein reconstituted upon interaction is a transcriptional activator (Fields and Song, 1989) have also been adapted for plant cell assays (Ehlert et al., 2006). In this case, the two-hybrid translational fusions are expressed transiently, for example, in polyethylene glycol-Ca²⁺-transfected protoplasts, from two independent Gateway expression clones and under the control of the 35S CaMV promoter. The tags code either for the DNA-binding domain or the activation domain of GAL4, and include a nuclear localization signal. The interaction reporter is encoded in a third cotransfected construct in which a promoter containing four GAL4-binding sites controls the transcription of the GUS enzyme (Table II, nos. 21–23).

Gene Stacking

It can be difficult to combine multiple transgenes in a single plant. MultiSite Gateway recombinational cloning can help solve this bottleneck. Binary destination vectors have been created for the expression of two or three genes under the control of different strong plant promoters (Table I, nos. 7 and 8). Before assembling the final expression vector, each target gene must be first captured in a separate entry clone (namely attL1-gene1-attL2, attL4-gene2-attL3, or attL6-gene3-attL5) and match a distinct Gateway destination cassette. The two or three destination cassettes are all located in the T-DNA region of a plant binary destination vector and are each flanked by different promoter and terminator regulatory sequences (Karimi et al., 2007).

Another vector system has been designed for the addition of multiple transgenes into the same binary destination vector via successive rounds of LR recombinations involving two types of entry clones (Chen et al., 2006). It takes advantage of different pairs of attL and attR sites, and of two alternating negative bacterial selection markers (ccdB and sacB). In this system, each added segment bringing in a new transgene also carries along the Gateway cassette where the next insert is recombined in the following round, in a scheme reminiscent of Matryoshka nesting dolls.

	VIRTUAL RECOMBINATION

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Several software packages offer tools that help plan Gateway cloning in silico (for review, see Katzen, 2007). Among them, only Vector NTI (version 9 and up) supports MultiSite Gateway (http://www.invitrogen.com/vntigateway). Its modules include (1) the optimal design of PCR hybrid primers (including attB and gene-specific sequences) for amplicon synthesis; (2) the creation of an entry clone in a BP reaction capturing an attB amplicon in an appropriate pDONR vector (from the VNTI molecule database); and (3) the creation of an expression clone resulting from a simple or a MultiSite LR clonase reaction, based on the recombined entry clone(s) and destination vector. All virtually generated molecules are documented as sequence files and maps with the annotated fragments.

	GENOME-SCALE RESOURCES COMPATIBLE WITH GATEWAY RECOMBINATIONAL CLONING

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Reference entry clones encoding well-documented genetic elements are highly valuable because the same accessions shared across multiple laboratories can be used recurrently in any assay for which adequate destination vectors exist. The Gateway system is particularly suited for systematic cloning projects because the highly specific BP and LR recombinations rely on relatively long sites of 21 to 232 bp (unlikely to occur by chance) and can therefore be performed regardless of the sequence of the transferred fragment(s). Furthermore, it is sufficiently robust for high-throughput and automated protocols.

Several large-scale clone collections have already been produced containing catalogued Arabidopsis genetic elements captured in Gateway clones (for review, see Hilson, 2006; Multinational Arabidopsis Steering Committee, 2007). They include cDNAs and ORFs in which the original stop codon is either maintained (closed configuration) or removed (open configuration) to enable the addition of a sequence coding for an in-frame C-terminal tag, and gene-specific sequence tags used for silencing (Hilson et al., 2004; Sclep et al., 2007). Collections of Arabidopsis promoters (Lee et al., 2006; http://www.psb.ugent.be/SAP/) and amiRNAs (http://2010.cshl.edu/) are or will soon be available in a Gateway-compatible format, as well as similar resources built on the genome sequence of other plant species.

	CONCLUSION

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Since the generation of thousands of constructs is not an insurmountable task anymore, researchers can envisage novel functional assays based on cloned sequence repertoires. Industrial laboratories have already developed genetic screens taking advantage of the modularity and flexibility of the Gateway cloning system. Another example of potential applications is a Gateway cassette including well-characterized, seed-specific regulatory sequences for the high-level production of antibodies in plant seeds (Van Droogenbroeck et al., 2007; M. Karimi and A. Depicker, unpublished data). However, to our knowledge, no regulatory body has yet ruled on the biosafety of recombination att site sequences in transgenes for the creation of genetically modified organisms. The Gateway system is commercialized for research purposes only and is considered by representatives of the agbiotech industry involved in transgenic crop improvement programs strictly as a research tool, not as a means toward product development.

Until now, an important bottleneck remains the stable transformation of many constructs into plants. For certain studies, such as transcriptional transactivation, subcellular localization, or plant two-hybrid surveys, assays in stably or transiently transformed cultured cells have proven very useful to accelerate genetic screens (e.g. Lurin et al., 2004; De Sutter et al., 2005; Koroleva et al., 2005; Ehlert et al., 2006; Goodin et al., 2007).

Besides assays in plants, many studies are also conducted in alternative heterologous systems for which Gateway destination vectors are continuously being developed, such as yeast two-hybrid and one-hybrid screens to decipher protein interactomes (Walhout et al., 2000; de Folter et al., 2005) and transcriptional networks (Deplancke et al., 2006), in vitro protein synthesis for the fabrication of multipurpose protein arrays (Ramachandran et al., 2004) or, more classically, synthesis in microbial organisms for the production of proteins in large amounts necessary for enzymological or crystallographic analyses. Of course, the same genetic elements isolated for plant assays might also serve in other applications, and vice versa, to understand the functional relationships between the many molecular entities making up a plant cell.

Considering the recent development of plant transformation vectors and large-scale clone resources, plant researchers have adopted the Gateway cloning system. In the foreseeable future, this platform will remain an important asset in projects requiring systematic cloning, modular assembly, and expression in various contexts. To take full advantage of the versatility of the system, biologists planning experiments should verify whether the DNA fragments they need are not already available as Gateway entry clones. In this context, well-funded and stable reference stock centers are essential to promote the exploitation of shared resources built in a common format.

	ACKNOWLEDGMENTS

 
We thank Rudy Vanderhaeghen and Annick Bleys for critical comments and Martine De Cock for help in preparing the manuscript.

Received August 6, 2007; accepted October 2, 2007; published December 6, 2007.

	FOOTNOTES

 
¹ This work was supported by two projects funded within the 6th European Framework Programme: EU-SOL (grant no. PL 016214–2 EU–SOL) and GENINTEG (grant no. LSHG–CT–2003–503303).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Pierre Hilson (pierre.hilson{at}psb.ugent.be).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.106989

^* Corresponding author; e-mail pierre.hilson{at}psb.ugent.be.

	LITERATURE CITED

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